Activatable Fluorophores for Imaging Immune Cell Function

Abstract

Conspectus Optical imaging has become an essential tool to study biomolecular processes in live systems with unprecedented spatial resolution. New fluorescent technologies and advances in optical microscopy have revolutionized the ways in which we can study immune cells in real time. For example, activatable fluorophores that emit signals after target recognition have enabled direct imaging of immune cell function with enhanced readouts and minimal background. In this Account, we summarize recent advances in the chemical synthesis and implementation of activatable fluorescent probes to monitor the activity and the role of immune cells in different pathological processes, from infection to inflammatory diseases or cancer. In addition to the contributions that our group has made to this field, we review the most relevant literature disclosed over the past decade, providing examples of different activatable architectures and their application in diagnostics and drug discovery. This Account covers the imaging of the three major cell types in the immune system, that is, neutrophils, macrophages, and lymphocytes. Attracted by the tunability and target specificity of peptides, many groups have designed strategies based on fluorogenic peptides whose fluorescence emission is regulated by the reaction with enzymes (e.g., MMPs, cathepsins, granzymes), or through Förster resonance energy transfer (FRET) mechanisms. Selective imaging of immune cells has been also achieved by targeting different intracellular metabolic routes, such as lipid biogenesis. Other approaches involve the implementation of diversity-oriented fluorescence libraries or the use of environmentally sensitive fluorescent scaffolds (e.g., molecular rotors). Our group has made important progress by constructing probes to image metastasis-associated macrophages in tumors, apoptotic neutrophils, or cytotoxic natural killer (NK) cells against cancer cells, among other examples. The chemical probes covered in this Account have been successfully validated in vitro in cell culture systems, and in vivo in relevant models of inflammation and cancer. Overall, the range of chemical structures and activation mechanisms reported to sense immune cell function is remarkable. However, the emergence of new strategies based on new molecular targets or activatable mechanisms that are yet to be discovered will open the door to track unexplored roles of immune cells in different biological systems. We anticipate that upcoming generations of activatable probes will find applications in the clinic to help assessing immunotherapies and advance precision medicine. We hope that this Account will evoke new ideas and innovative work in the design of fluorescent probes for imaging cell function.

Emerging Biomaterials Imaging Antitumor Immune Response

“…The goal of using imaging techniques for predicting immunotherapy is to provide patients with personalized treatment regimens, but there remain some obstacles to identifying the characteristics of an effective anticancer immune response owing to the complexity of the immune microenvironment and patient heterogeneity. [ 330 ] The combination of multiple tracers or imaging modalities can help monitor or predict immunotherapy responses more effectively, but it can incur high costs. Therefore, it remains challenging to predict immunotherapy responses in an efficient, cost‐effective, and patient‐friendly manner.…”

Dihydroxanthene-Based Near-infrared Fluorescent Probes for Monitoring Mitochondrial Viscosity in Living Cells and Mice

“…Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. 1 H NMR spectra were obtained at 400 MHz using Bruker NMR spectrometers, and 13 C NMR spectra were recorded at 100 MHz. The mass spectra were obtained using the Bruker maXis ultrahigh-resolution-TOF MS system.…”

“…Purification by flash chromatography on silica gel (methylene dichloride/methanol = 15:1, v/v) afforded 145 mg of DHX-V-3 (26% yield). 1…”

“…The catalyst was filtered off, then washed with acetonitrile to yield dark green solid (129 mg, 63%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.15 (s, 1H), 8.71 (d, J = 6.8 Hz, 2H), 8.22 (d, J = 15.7 Hz, 1H), 8.07 (d, J = 6.8 Hz, 2H), 7.12 (d, J = 8.3 Hz, 1H), 6.76 (d, J = 2.5 Hz, 2H), 6.65−6.52 (m, 2H), 4.44 (q, J = 7.2 Hz, 2H), 2.60−2.37 (m, 7H), 1.74 (p, J = 5.9 Hz, 2H), 1.50 (t, J = 7.2 Hz, 3H). 13 Synthesis of DHX-V-2.…”

“…To a solution of compound 2 37 (50 mg, 0.17 mmol) and 4-ethyl-1-methyl-pyridin-1-ium (66 mg, 0.26 mmol) in anhydrous acetonitrile (5 mL) was added an amount of piperidine, the mixture was stirred under an argon atmosphere at 70 °C for 7 h. The resulting mixture was allowed to cool to room temperature, and the precipitate was filtered off and then washed with acetonitrile to yield dark green solid (53 mg, 61%). 1 H NMR (400 MHz, chloroform-d) δ 8.68 (d, J = 6.7 Hz, 2H), 8.15 (d, J = 15.3 Hz, 1H), 7.67 (d, J = 6.5 Hz, 2H), 6.96 (d, J = 8.7 Hz, 1H), 6.57 (s, 1H), 6.53− 6.45 (m, 1H), 6.40 (dd, J = 8.9, 2.4 Hz, 1H), 6.16 (d, J = 15.2 Hz, 1H), 4.59 (q, J = 7.3 Hz, 2H), 3.44 (q, J = 7.0 Hz, 4H), 2.48 (dt, J = 23.7, 6.2 Hz, 4H), 1.78 (p, J = 6.0 Hz, 2H), 1.68 (s, 1H), 1.59 (t, J = 7.3 Hz, 3H), 1.22 (t, J = 7.0 Hz, 6H). 13 Synthesis of Compound 3.…”